1. Field of the Invention
The present invention relates generally to cell culture medium formulations. Specifically, the present invention provides systems comprising defined cell culture medium formulations that facilitate the in vitro cultivation of epithelial cells, particularly keratinocytes. The present invention also provides methods for cultivation of animal cells using these systems.
2. Related Art
Cell culture media provide the nutrients necessary to maintain and grow cells in a controlled, artificial and in vitro environment. Characteristics and compositions of the cell culture media vary depending on the particular cellular requirements. Important parameters include osmolarity, pH, and nutrient formulations.
Media formulations have been used to cultivate a number of cell types including animal, plant and bacterial cells. Cells cultivated in culture media catabolize available nutrients and produce useful biological substances such as monoclonal antibodies, hormones, growth factors and the like. Such products have therapeutic applications and, with the advent of recombinant DNA technology, cells can be engineered to produce large quantities of these products. Thus, the ability to cultivate cells in vitro is not only important for the study of cell physiology, but is also necessary for the production of useful substances which may not otherwise be obtained by cost-effective means.
Cell culture media formulations have been well documented in the literature and a number of media are commercially available. In early cell culture work, media formulations were based upon the chemical composition and physicochemical properties (e.g., osmolality, pH, etc.) of blood and were referred to as xe2x80x9cphysiological solutionsxe2x80x9d (Ringer, S., J. Physiol., 3:380-393 (1880); Waymouth, C., In: Cells and Tissues in Culture, Vol. 1, Academic Press, London, pp. 99-142 (1965);Waymouth, C., In Vitro 6:109-127 (1970)). However, cells in different tissues of the mammalian body are exposed to different microenvironments with respect to oxygen/carbon dioxide partial pressure and concentrations of nutrients, vitamins, and trace elements; accordingly, successful in vitro culture of different cell types will often require the use of different media formulations. Typical components of cell culture media include amino acids, organic and inorganic salts, vitamins, trace metals, sugars, lipids and nucleic acids, the types and amounts of which may vary depending upon the particular requirements of a given cell or tissue type.
Typically, cell culture media formulations are supplemented with a range of additives, including undefined components such as fetal bovine serum (FBS) (10-20% v/v) or extracts from animal embryos, organs or glands (0.5-10% v/v). While FBS is the most commonly applied supplement in animal cell culture media, other serum sources are also routinely used, including newborn calf, horse and human. Organs or glands that have been used to prepare extracts for the supplementation of culture media include submaxillary gland (Cohen, S., J. Biol. Chem. 237:1555-1565 (1961)), pituitary (Peehl, D. M., and Ham, R. G., In Vitro 16:516-525 (1980); U.S. Pat. No. 4,673,649), hypothalamus (Maciag, T., et al., Proc. Natl. Acad. Sci. USA 76:5674-5678 (1979); Gilchrest, B. A., et al., J. Cell. Physiol. 120:377-383 (1984)), ocular retina (Barretault, D., et al., Differentiation 18:29-42 (1981)) and brain (Maciag, T., et al., Science 211:1452-1454 (1981)). These types of chemically undefined supplements serve several useful functions in cell culture media (Lambert, K. J. et al., In: Animal Cell Biotechnology, Vol. 1, Spier, R. E. et al., Eds., Academic Press New York, pp. 85-122 (1985)). For example, these supplements provide carriers or chelators for labile or water-insoluble nutrients; bind and neutralize toxic moieties; provide hormones and growth factors, protease inhibitors and essential, often unidentified or undefined low molecular weight nutrients; and protect cells from physical stress and damage. Thus, serum or organ/gland extracts are commonly used as relatively low-cost supplements to provide an optimal culture medium for the cultivation of animal cells.
Unfortunately, the use of serum or organ/gland extracts in tissue culture applications has several drawbacks (Lambert, K. J. et al., In: Animal Cell Biotechnology, Vol 1, Spier, R. E. et al., Eds., Academic Pres New York, pp. 85-122 (1985)). For example, the chemical composition of these supplements may vary between lots, even from a single manufacturer. The supplements may also be contaminated with infectious agents (e.g., mycoplasma and viruses) which can seriously undermine the health of the cultured cells when these contaminated supplements are used in cell culture media formulations. Cell surface chemistry, which is a critical portion of the in vitro microenvironment for many cell types, can be adversely modified via adsorption or incorporation of serum or extract proteins. The use of undefined components such as serum or animal extracts also prevents the true definition and elucidation of the nutritional and hormonal requirements of the cultured cells, thus eliminating the ability to study, in a controlled way, the effect of specific growth factors or nutrients on cell growth and differentiation in culture. Moreover, undefined supplements prevent the researcher from studying aberrant growth and differentiation and the disease-related changes in cultured cells. Finally and most importantly to those employing cell culture media in the industrial production of biological substances, serum and organ/gland extract supplementation of culture media can complicate and increase the costs of the purification of the desired substances from the culture media due to nonspecific co-purification of serum or extract proteins.
To overcome these drawbacks of the use of serum or organ/gland extracts, a number of so-called xe2x80x9cdefinedxe2x80x9d media have been developed. These media, which often are specifically formulated to support the culture of a single cell type, contain no undefined supplements and instead incorporate defined quantities of purified growth factors, proteins, lipoproteins and other substances usually provided by the serum or extract supplement. Since the components (and concentrations thereof) in such culture media are precisely known, these media are generally referred to as xe2x80x9cdefined culture media.xe2x80x9d Often used interchangeably with xe2x80x9cdefined culture mediaxe2x80x9d is the term xe2x80x9cserum-free mediaxe2x80x9d or xe2x80x9cSFM.xe2x80x9d A number of SFM formulations are commercially available, such as those designed to support the culture of endothelial cells, keratinocytes, monocytes/macrophages, fibroblasts, chondrocytes or hepatocytes which are available from GIBCO/LTI (Gaithersburg, Md.). The distinction between SFM and defined media, however, is that SFM are media devoid of serum, but not necessarily of other undefined components such as organ/gland extracts. Indeed, several SFM that have been reported or that are available commercially contain such undefined components, including several formulations supporting in vitro culture of keratinocytes (Boyce, S. T., and Ham, R. G., J. Invest. Dermatol. 81:33 (1983); Wille, J. J., et al., J. Cell. Physiol. 121:31 (1984); Pittelkow, M. R., and Scott, R. E., Mayo Clin. Proc. 61:771 (1986); Pirisi, L., et al., J. Virol. 61:1061 (1987); Shipley, G. D., and Pittelkow, M. R., Arch. Dermatol. 123:1541 (1987); Shipley, G. D., et al., J. Cell. Physiol. 138:511-518 (1989); Daley, J. P., et al., FOCUS (GIBCO/LTI) 12:68 (1990); U.S. Pat. Nos. 4,673,649 and 4,940,666). SFM thus cannot be considered to be defined media in the true definition of the term.
Defined media generally provide several distinct advantages to the user. For example, the use of defined media facilitates the investigation of the effects of a specific growth factor or other medium component on cellular physiology, which may be masked when the cells are cultivated in serum- or extract-containing media. In addition, defined media typically contain much lower quantities of protein (indeed, defined media are often termed xe2x80x9clow protein mediaxe2x80x9d) than those containing serum or extracts, rendering purification of biological substances produced by cells cultured in defined media far simpler and more cost-effective.
Some extremely simple defined media, which consist essentially of vitamins, amino acids, organic and inorganic salts and buffers have been used for cell culture. Such media (often called xe2x80x9cbasal mediaxe2x80x9d), however, are usually seriously deficient in the nutritional content required by most animal cells. Accordingly, most defined media incorporate into the basal media additional components to make the media more nutritionally complex, but to maintain the serum-free and low protein content of the media. Examples of such components include serum albumin from bovine (BSA) or human (HSA); certain growth factors derived from natural (animal) or recombinant sources such as EGF or FGF; lipids such as fatty acids, sterols and phospholipids; lipid derivatives and complexes such as phosphoethanolamine, ethanolamine and lipoproteins; protein and steroid hormones such as insulin, hydrocortisone and progesterone; nucleotide precursors; and certain trace elements (reviewed by Waymouth, C., in: Cell Culture Methods for Molecular and Cell Biology, Vol. 1: Methods for Preparation of Media, Supplements, and Substrata for Serum-Free Animal Cell Culture, Barnes, D. W., et al., eds., New York: Alan R. Liss, Inc., pp. 23-68 (1984), and by Gospodarowicz, D., Id., at pp 69-86 (1984)).
The epithelium lines the internal and external surfaces of the organs and glands of higher organisms. Because of this localization at the external interface between the environment and the organism (e.g., the skin) or at the internal interface between an organ and the interstitial space (e.g., the intestinal mucosal lining), the epithelium has a major role in the maintenance of homeostasis. The epithelium carries out this function, for example, by regulating transport and permeability of nutrients and wastes (Freshney, R. I., in: Culture of Epithelial Cells, Freshney, R. I., ed., New York: Wiley-Liss, pp. 1-23 (1992)).
The cells making up the epithelium are generically termed epithelial cells. These cells may be present in multiple layers as in the skin, or in a single layer as in the lung alveoli. As might be expected, the structure, function and physiology of epithelial cells are often tissue-specific. For example, the epidermal epithelial cells of the skin are organized as stratified squamous epithelium and are primarily involved in forming a protective barrier for the organism, while the secretory epithelial cells of many glands are often found in single layers of cuboidal cells that have a major role in producing secretory proteins and glycoproteins. Regardless of their location or function, however, epithelial cells are usually regenerative. That is, under normal conditions, or in response to injury or other activating stimulus, epithelial cells are capable of dividing or growing. This regenerative capacity has facilitated the in vitro manipulation of epithelial cells, to the point where a variety of primary epithelial cells and cell lines have been successfully cultivated in vitro (Freshney, Id).
The specialized epithelial cells found in the epidermis of the skin are known as keratinocytes. In the upper, cornified layers of the skin (those exposed to the environment), the cytoplasm of the keratinocytes is completely replaced with keratin and the cells are dead. The keratinocytes located in the lower layers, however, particularly in the basal epidermis (stratum basale), actively divide and ultimately migrate up through the more superficial layers to replace those cells being sloughed off at the external surface. Accordingly, the skin can be thought of as a dynamic organ comprising keratinocytes that are constantly dividing, maturing and ultimately dying.
Cultures of human keratinocytes are increasingly being used in examinations of skin structure and disease, and as in vitro models of human skin in toxicology studies (Boyce, S. T., and Ham, R. G., in: In Vitro Models for Cancer Research, vol. III, Webber, M. M., et al., eds., Boca Raton, Fla.: CRC Press, Inc., pp. 245-274 (1985)). Successful culture of keratinocytes has proven, however, to be somewhat difficult, owing primarily to their nutritional fastidiousness (Gilchrest, B. A., et al., J. Cell. Physiol. 120:377-383 (1984)). For example, in most early studies using traditional serum-supplemented culture media, keratinocytes from skin explants were rapidly overgrown by less fastidious and faster-growing fibroblasts that were also resident in the tissue (Freshney, Id.). Thus, there has been substantial work expended in the attempt to formulate culture media favoring the selection and successful in vitro cultivation of human keratinocytes.
A variety of systems have been developed to culture human keratinocytes. Early work in this area used specialized culture media such as Medium 199 (Marcelo, C. L., et al., J. Cell Biol. 79:356 (1978)) and NCTC 168 (Price, F. M., et al., In Vitro 16:147 (1980)) supplemented with serum. Alternatively, keratinocyte growth and colony formation have been shown to be improved by plating cells on lethally irradiated 3T3 fibroblasts and by adding epidermal growth factor (EGF) and hydrocortisone to the medium (Rheinwald, J. G., and Green, H., Cell 6:331 (1975)). One of the first serum-free medium formulations developed for keratinocyte culture was based on Medium 199 and included a growth factor cocktail comprising bovine brain extract (Gilchrest, B. A., et al., J. Cell. Physiol. 112:197 (1982)), and serum-free culture of human keratinocytes without the use of 3T3 fibroblast feeder layers became widely accepted upon the development of a more specialized basal medium, MCDB-153 (Boyce, S. T., and Ham, R. G., J. Invest. Dermatol. 81:33 (1983); U.S. Pat. Nos. 4,673,649 and 4,940,666). Serum-free MCDB-153 includes trace elements, ethanolamine, phosphoethanolamine, hydrocortisone, EGF, and bovine pituitary extract (BPE). This medium and several enhanced versions have been used widely for human keratinocyte cultivation (Pittelkow, M. R., and Scott, R. E., Mayo Clin. Proc. 61:771 (1986); Pirisi, L., et al., J. Virol. 61:1061 (1987); Shipley, G. D., and Pittelkow, M. R., Arch. Dermatol. 123:1541 (1987); Daley, J. P., et al., FOCUS (GIBCO/LTI) 12:68 (1990)). The use of BPE is also common to many commercially available media for keratinocyte cultivation, including KGM (Clonetics Corporation; San Diego, Calif.), CS-2.0 Keratinocyte Cell Growth Medium (Cell Systems, Inc.; Kirkland, Wash.), M154 (Cascade Biologicals, Inc.; Portland, Oreg.) and Keratinocyte-SFM (GIBCO/LTI; Gaithersburg, Md.).
Serum-free medium containing BPE as the primary mitogen, however, has several drawbacks, as generally described above. For example, the undefined composition of BPE complicates experimental models and interpretation of results, and may either stimulate or inhibit the growth or differentiation of keratinocyte cultures, depending on the concentrations of other components in the medium (Wille, J. J., et al., J. Cell. Physiol. 121:31 (1984)). In addition, BPE requires titration in different cell systems, and its stability in medium is limited to about four weeks under normal use and storage conditions. There has been at least one report of a fully defined medium for the culture of epidermal cells, wherein BPE is replaced with epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1) and increased quantities of six specific amino acids (U.S. Pat. No. 5,292,655). However, this medium was designed for the specific purpose of in vitro formation of a skin substitute comprising differentiated keratinocytes, and may not be ideal for supporting continuous cultures of actively growing cells.
Thus, a need remains for defined culture media, that are serum- and organ/gland extract-free, for the cultivation of animal epithelial cells including keratinocytes. Such culture media will facilitate studies of the effects of growth factors and other stimuli on cellular physiology, will allow easier and more cost-effective purification of biological substances produced by cultured animal cells in the biotechnology industry, and will provide more consistent results in methods employing the cultivation of animal epithelial cells. The current invention provides such defined media.
The present invention provides defined culture media that replace BPE with growth-promoting additives such as insulin, EGF and other additives. Specifically, the invention provides a cell culture medium, capable of supporting the cultivation of an animal epithelial cell in vitro, comprising insulin, EGF, and at least two additional additives from the group consisting of FGF, an agent that increases intracellular levels of cyclic adenosine monophosphate (cAMP) and ascorbic acid. The medium provided by the present invention may be a 1xc3x97formulation, or may be concentrated as a 10xc3x97 or higher formulation. The basal medium of the present invention comprises a number of ingredients, including amino acids, vitamins, organic and inorganic salts, sugars and other components, each ingredient being present in an amount which supports the cultivation of an animal epithelial cell in vitro. The medium may be used to culture a variety of animal epithelial cells, including primary cells (e.g., keratinocytes or cervical epithelial cells) and established cell lines (e.g., HeLa cells). Cells supported by the medium of the present invention may be derived from any animal, preferably a mammal, and most preferably a human. The present invention also provides methods of culturing animal epithelial cells using the culture medium formulations disclosed herein, comprising the steps of (a) contacting an animal cell with the cell culture medium of the present invention; and (b) cultivating the animal cell under conditions suitable to support its cultivation in vitro. The invention also provides kits for use in the cultivation of an animal epithelial cell. Kits according to the present invention comprise a carrier means having in close confinement therein one or more container means, wherein a first container means contains a basal culture medium as described above, a second carrier means contains a insulin, a third container means contains EGF, a fourth container means contains FGF, a fifth container means contains at least one agent that increases intracellular levels of cAMP, a sixth container means contains heparin and a seventh container means contains ascorbic acid. In a preferred embodiment, the second container means of the kits contains insulin, EGF, FGF, at least one agent that increases intracellular levels of cAMP, heparin and ascorbic acid together in admixture. The invention further provides cell culture compositions comprising the culture media of the present invention and an animal epithelial cell. The invention also provides compositions comprising heparin, EGF, FGF, at least one agent that increases intracellular levels of cAMP, and optionally ascorbic acid, which compositions may be used to replace organ or gland extracts in serum-free animal cell culture media. The culture media of the present invention are suitable for use in the isolation and initiation of primary epithelial cell cultures, as well as for the expansion of established epithelial cell cultures. Additionally, the media of the present invention provide superior growth, and maintenance of morphological and physiological markers, of primary animal epithelial cells.